Alzheimer's disease (AD) is a progressive neurodegenerative disorder that develops in aged individuals and causes loss of memory and cognitive processes. The world-wide cost of AD is estimated to be $315.4 billion annually, which is greater than the budget of all but eight of the world's countries. There is currently no means of stopping the progression of AD. A central hypothesis is that the abnormal accumulation of beta-amyloid (A2) peptides causes neuronal degeneration in brain regions responsible for memory. A potential means of stopping, or possibly reversing, the progression of the disease is to reduce A2 production by inhibiting the enzymes that produce it. A particularly attractive target for doing this is the 2-secretase, which cleaves the amyloid precursor protein (APP) to produce the amino terminal end of A2. We have found that the compounds, E64d and CA074Me are efficacious in AD animal models expressing APP containing the wild-type 2-secretase site, which is found in most AD patients. Significantly, we discovered that E64d and CA074Me improve memory and reduce brain amyloid plaques, both characteristics of AD, in transgenic mice expressing human APP containing the wild-type 2-secretase site. They also reduced brain A2 and CTF2, a fragment produced by 2-secretase cleavage, which suggests that the compounds act by inhibiting brain 2-secretase activity. E64d and CA074Me are the ethyl and methyl ester prodrugs, respectively, of their acid forms, E64c and CA074, which are the active in vivo agents. Although structurally very similar, E64c inhibits cysteine proteases generally whereas CA074 selectively inhibits the cysteine protease, cathepsin B. E64d was found clinically safe to use when first developed for muscular dystrophy but was discontinued due to lack of efficacy. CA074 is expected to have an even safer profile because it is a cathepsin B specific inhibitor Thus, E64c and CA074 are likely to be both efficacious and safe to use for AD. New E64c and CA074 prodrugs are needed that increase the brain concentrations after oral administration. This grant will design and synthesize two new classes of such prodrugs and evaluate them in cell and AD animal models. The first will be to make E64c and CA074 prodrugs containing esters known to increase the lipophilicity and increase the likelihood of brain penetration. The other is E64c- and CA074-trigonelline compounds, which increase the brain-to- systemic dose ratio by trapping the drug in the brain while facilitating its systemic elimination. The efficacy of orally administering the prodrugs will be evaluated in normal and transgenic AD animal models. This project promises to have a major impact on AD drug translational research by ushering in an entirely new class of cysteine protease inhibitor AD drugs. PUBLIC HEALTH RELEVANCE: The project is relevant to the public health. Specifically, this project will develop new and effective Alzheimer's disease drugs. Currently, there is no effective means of stopping the progress of this devastating disease and there is an urgent need for new drugs that do so. This project will result in drugs that may halt or, possibly, reverse the progression of the disease. As such, the project has enormous potential for improving the lives of millions of Americans and their families.